ChIP-Atlas: SRX8819500

Antigen

Cell type Class: Pluripotent stem cell
Cell type: Embryoid Bodies
MeSH Description: Spontaneous aggregations of human embryonic stem cells that occur in vitro after culturing in a medium that lacks LEUKEMIC INHIBITORY FACTOR. The embryoid bodies can further differentiate into cells that represent different lineages.

library_strategy: ChIP-Seq
library_source: GENOMIC
library_selection: ChIP
library_construction_protocol: Cells were cross-linked with 1 % formaldehyde for 10 min, followed by quenching with 0.125 M glycine, harvesting and washing in PBS containing protease inhibitors. The cells were sequentially lysed in three buffers (lysis buffer 1: 50 mM HEPES, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% TX-100; lysis buffer 2: 10 mM Tris, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA; lysis Buffer 3: 10 mM Tris, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine) with rotation for 10 min in between. Then, the chromatin was sonicated with the Epishear™ Probe Sonicator (Active Motif) with 20 s ON and 30 s OFF for 8 cycles. After centrifugation, the supernatant was divided into input and ChIP samples. The ChIP samples were incubated with specific antibodies (Supplementary Table 4) overnight, followed by immunoprecipitation with Dynabeads Protein G beads (invitrogen). Next, the beads were washed with RIPA buffer (50 mM Hepes, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% Na-Deoxycholate) on a magnet, eluted (50 mM Tris, 10 mM EDTA, 1% SDS), and reverse crosslinked at 65 °C overnight in parallel with the input. Finally, DNAs were purified with the ChIP DNA Clean & Concentrator (Zymo Research). Illumina TruSeq